Table 1.

Summary of modifications to RT-qPCR components for efficient multiplex amplification.

RT-PCR component	Modification	Singleplex	Multiplex
Binding protein gp2.5	The concentration of binding protein was raised to achieve 0.2 µg per pmol of primers and probes to sequester the additional amount of primer pairs and TaqMan probes in multiplex reaction	0.51 µg/µL	0.95 µg/µL
Taq polymerase	The quantity of Taq polymerase was increased for efficient amplification of multiple targets54,56	0.3 U/µL	0.6 U/µL
KCl	KCl facilitates annealing of DNA molecules and increase in KCl increases product yield of shorter amplicons55	55 mM	90 mM
MgCl2	Facilitates annealing of DNA molecules and acts as a cofactor of Taq polymerase51	2.0 mM	2.6 mM
dNTP	More dNTPs are required to produce more amplification products. As increasing amounts of dNTPs can bind to Mg2+ electrostatically, reduce the amount of Mg2+ available and inhibit the reaction55, concentrations of MgCl2 and dNTPs were tested in a factorial experimental design	400 μM	500 μM
Primers and Taqman probes	Primers and Taqman probes were redesigned and verified by gel electrophoresis to minimize dimer formation and off-target amplification	See Supplementary Table S1 for sequences	See Supplementary Table S1 for sequences